Muscarinic Acetylcholine Type 1 Receptor Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons: A Phenotype Rescued by Antagonist Treatment
Bibliographic record
Abstract
Objective The muscarinic acetylcholine type 1 receptor (M 1 R) is a metabotropic G protein coupled receptor. Knockout of M 1 R or exposure to selective or specific receptor antagonists elevates neurite outgrowth in adult sensory neurons and blockade of associated cholinergic pathways is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M 1 R activation constrained neurite outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking and that this could be rescued by antagonist treatment. Methodology We overexpressed M 1 R and M 1 R DREADD ( d esigner r eceptors e xclusively a ctivated by d esigner d rugs) mutant in primary cultures of adult rat dorsal root ganglion (DRG) neurons and in tumor cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and neurite outgrowth. The M 1 R‐DREADD mutant contained two mutations of conserved orthosteric site residues that cause minimal responsiveness to ACh. In addition, we used CRISPR/Cas9 based Gα 12/13 knockout HEK293 cells and siRNA mediated knockdown of Gα 13 in cultured DRG neurons to study M 1 R Gα 13 protein mediated tubulin cytoskeletal dynamics. No M 1 R ligand was added to the culture media. Results Overexpression of M 1 R by adult sensory neurons caused dissolution of the polymerized tubulin cytoskeleton, but not the actin network and significantly reduced neurite outgrowth. Conversely, over‐expression of a M 1 R‐DREADD mutant increased neurite outgrowth. These data suggest that endogenously released M 1 R ligand interacts with the M 1 R to suppress neurite outgrowth. M 1 R‐dependent disruption of the cytoskeleton significantly diminished mitochondrial abundance and trafficking in the distal neurites. Treatment with the specific M 1 R antagonist – muscarinic toxin 7 (MT7, 100 nM) or the selective M 1 R antagonist pirenzepine (1 μM) rescued cytoskeletal abnormalities caused by M 1 R overexpression by sequestering G‐proteins (Gα 12/13 ) to the receptor. In addition, Gα 12/13 null HEK293 cells exhibited increased polymerized tubulin and knockdown of Gα 13 protected from tubulin cytoskeletal destabilization and restored growth in neurons overexpressing the M 1 R. Conclusions Our study indicates that constitutively high expression of M 1 R destabilizes cytoskeletal dynamics through activation of Gα 13 protein that increased tubulin de‐polymerization, impeded mitochondrial transport and reduced neurite outgrowth. This suggests a novel mechanism of M 1 R control of G protein‐dependent (Gα13) modulation of the tubulin cytoskeleton, mitochondrial trafficking and neurite outgrowth in axons of adult sensory neurons. Manipulation of this novel pathway could be used to treat dying‐back neuropathies since anti‐muscarinic drugs are currently utilized for other clinical conditions. Support or Funding Information Supported by CIHR grant # MOP‐130282 (PF) and NIH grant NS081082 (NAC) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".